Wounds can be divided into two major categories: acute wounds, such as those associated with surgical incisions and excisions, bites, burns, cuts and abrasions, as well as more traumatic wounds such as lacerations and those caused by crush or gun shot injuries, and chronic or impaired non-healing wounds, such as those associated with diabetes, venous and arterial stasis leg ulcers, foot ulcers, and pressure sores to name a few.
Acute wound healing has been categorized into four phases: coagulation, inflammation, proliferation, and remodeling (Singer, A. J. and Clark, R. A. (1999) N. Engl. J. Med. 341:738-746). At the time of injury, coagulation is initiated by activated platelets binding thrombin and forming a plug. Vasoconstriction and cytokine release also occur. For example, platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), and transforming growth factor-beta (TGF-β) are common factors released. During the second phase, inflammatory cells, such as macrophages and polymorphonuclear (PMN) cells are recruited, which phagocytose (engulf) bacteria, remove dead tissue/cells (wound debridement), and produce additional cytokines and growth factors such as IL-6 and TGF-β. Fibroblasts are also recruited and produce matrix components such as fibronectin and collagen. Within 1-2 days after injury, keratinocytes proliferate at the wound margin and subsequently migrate both, over the wound and upward from any remaining hair follicles and sweat ducts to begin wound resurfacing, termed re-epithelialization. Matrix metalloproteinases (MMPs) are produced by inflammatory cells, and help prepare the wound for angiogenesis (new blood vessel formation). In the proliferation phase, fibroblasts and endothelial cells proliferate, and fibroblasts secrete extracellular matrix proteins forming granulation tissue. Later, fibroblasts remodel tissue, macrophages continue to debride the wound, fibroblasts continue to synthesize and release growth factors and extracellular matrix (ECM) proteins, such as collagens and fibronectin, and a subpopulation of fibroblasts differentiate into myofibroblasts that produce more ECM and cause wound contraction. The granulation tissue serves as a matrix over which the keratinocytes migrate to create the new epidermal surface across the wound. The wound contracts and later a scar is formed by excessive tissue remodeling. During the final remodeling phase, collagen fibrils in the scar are degraded by MMPs. Thus, numerous cell types and complex molecular events and biologic processes must stochastically interact to bring about [cutaneous] repair of injury. The most critical molecular events involved in normal wound healing are cell migration, cell proliferation, and wound contraction. The major cell types involved in the [cutaneous] wound healing process are keratinocytes, fibroblasts, endothelial cells, and immune cells, and mesenchymal stem cells. General tissue repair involves similar cellular processes in a more regenerative sense largely involving proliferation and differentiation of the particular cell types composing a damaged organ, bone, cartilage, tendon, ligament etc and angiogenesis to supply the repairing tissue with nutrients. The migration of mesenchymal stem cells endothelial stem cells are shown to be an important cell type involved in the wound healing and tissue repair process. Stem cells, normally involved in development, are released from the bone marrow when cytokines are released into the circulation upon injury. These progenitor cells, including mesenchymal stem cells (MSCs), fibrocytes (derived from MSCs; CD34+, Col I+, CD11b+, CD13+, MHC class II+), and endothelial progenitors cells (EPCs) provide important contributions to the wound healing/tissue repair and regeneration process (Liu, Z. J. et al (2009) J Cell Biochem. 106:984-991; Abe, R. et al (2001) J. Immunol. 166:7556-7562).
Unlike acute wound healing, chronic wound healing does not proceed normally through the four healing phases. Chronic wounds are characterized by a lack of continuity and integrity of healing with wounds lasting more than 8 weeks, no healing, or a recurring wound (Liu, Z. J. and Velasquez, O. C. (2008) Antiox. Redox. Signal. 10:1869-1882). These wounds are arrested in the inflammatory phase of healing and demonstrate persistent infection concomitant with a constant influx of neutrophils that perpetuate the release of cytotoxic enzymes, free oxygen radicals and other inflammatory mediators. There are increased levels of cytokines and continued destruction of tissue by matrix metalloproteinases (MMPs) (Singer, A. J. and Clark, R. A. (1999) N. Engl. J. Med. 341:738-746). Specifically, the inflammatory excess is characterized by excessive production of Interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), and MMPs). Other defects are a deficiency of important growth factors needed for proper healing, and bacterial overgrowth and senescence of fibroblasts. Further, the epithelial layer fails to cover the entire surface of the wound and, consequently, a chronic wound remains open and subject to infection. Bacteria colonize the chronic wound beneath a biofilm layer (which they secrete), activate virulence factors, and trigger NFκB-dependent inflammatory pathways, thereby continuing the process of inflammatory excess that prevents proper healing of the wound. A resulting dead tissue accumulates completely retarding healing and therefore, chronic wounds require frequent surgical debridement to remove debris. Failure of mesenchymal stem cells to home to injured sites is a problem in chronic wound healing leading to lack of proper cell differentiation and angiogenesis. It has been shown that chronic-impaired wounds, such as diabetic wounds, contain less stromal-derived factor (SDF-1α), a protein required for homing of EPCs to the wound.
A type of chronic wound is a diabetic wound, which are largely diabetic foot ulcers (DFUs). Similar to other chronic wounds but more severe, these wounds are defective in cell proliferation, the migration of cells into the wound including macrophage infiltration, extracellular matrix production, clearance of dead tissue and apoptotic cells, and fibromyoblast differentiation (Ochoa, O. et al (2007) Vasc 15:350-355). It is also proposed that high glucose levels (hyperglycemia) in diabetics cause cell wall rigidity, which impedes red blood cell permeability, and impairs blood flow through the microvasculature causing ischemia at the wound surface. New blood vessel growth is impaired by lack of VEGF production (Galiano, R. D. et al (2004) Am. J. Pathol. 161:19351947.
Novel therapies/agents to heal all types of chronic non-healing wounds and extensively injured tissue, including epidermal and dermal skin substitutes (cell-based therapies/wound devices) have largely failed causing an insurmountable and unsolved medical problem (Clark, R. A. et al (2007) J. Invest. Dermatol. 127:1018-1029). Existing pharmaceutical agents, such as Regranex® gel, are currently used to treat acute and chronic wounds. Regranex® gel contains becaplermin, a recombinant human platelet-derived growth factor isoform dimer, BB (PDGF-BB), which promotes cellular proliferation of the cells of the dermis, which are mainly fibroblasts, and angiogenesis. It is indicated for the treatment of lower extremity diabetic neuropathic ulcers that extend into the subcutaneous tissue or beyond and have an inadequate blood supply. An increased rate of malignancies and death in patients using Regranex® gel has been reported, indicating that safer alternatives to this drug are needed. Other proteins that have shown promise in vivo include vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), epidermal growth factor (EGF), transforming growth factor-β (TGF-β) and others. Galiano et al., Am J Pathol 2004, 164:1935-1947; Michaels et al., Wound Repair and Regeneration 2005, 13:506-512; Obara et al., Wound Repair and Regeneration 2005, 13:390-397; Greenhalgh et al., Am J Pathol 1990, 136:1235-1246; Acosta et al., International Wound Journal 2006, 3:232-239.
A role for calreticulin, a 46 kDa protein (it resolves at a higher molecular weight in SDS-page, e.g., 55-60) associated with hyaluronan, in the treatment of acute wounds (and reduced scar formation), such as surgical wounds and wounds incurred in accidental trauma, has been described by the present inventors. See, e.g., U.S. Pat. No. 5,591,716. Calreticulin is a highly conserved major calcium-binding protein of the endoplasmic reticulum (ER) consisting of three structurally and functionally distinct domains—the N, P and the C domains, as shown in FIG. 36. (Bedard, K., et al., (2005) Int Rev Cytol 245, 91-121; Michalak, M., et al., (2009) Biochem J 417, 651-666) (FIGS. 37 and 38). As shown in FIGS. 36 and 37, the middle P and C-terminal domains contain a number of high- and low-affinity calcium interacting sites, respectively. The N-terminal domain contains a signal sequence for targeting to the ER and the C-terminal domain has a KDEL sequence at its C-terminus, for retrieval/retention in the ER. Within the lumen of the ER, CRT in concert with other ER-resident chaperones mainly, 1) ensures proper folding of proteins and glycoproteins mainly via its lectin-binding site, 2) prevents protein aggregation and 3) is engaged in protein quality control through identifying and banning misfolded proteins from the ER for ubiquitin-mediated destruction. Another important function for CRT directed from the ER is in the regulation of calcium metabolism, which influences a variety of cellular functions including cell signaling, particularly through integrins. The heralded functions of calreticulin are intracellular, in calcium homeostasis and in binding N-linked oligosaccharide protein intermediates to ensure proper glycoprotein conformation in the ER. Johnson, S. et al. (2001) Trends Cell Biol. 11:122-129; Bedard, K. et al. (2005) Int. Rev. Cytol. 11:122-129; Sezestakowska, D. et al. (2006) International Workshop on Calreticulin, Niagara Falls, Canada. 1:135-139; Gold, L. I. et al. (2006) J. Investig. Dermatol. Symp. Proc. 11:57-65. However, more recently, roles for calreticulin in extracellular functions have been emerging (FIG. 37) such as the processes of wound healing, adaptive immune response in cancer, clearance of apoptotic cells by phagocytes, thrombospondin-mediated migration and prevention from anoikis, and the uptake of necrotic tumor cells by dendritic cells (review Michalak, M. et al (2009) Biochem. J. 417:651-656).
Chronic wounds and their management are very different than acute wounds and, thus, therapeutic agents that are useful for the treatment of acute wounds may not be as useful for the treatment of chronic wounds. Thus, there remains a need to discover new therapeutic agents and methods of treatment that are useful for the healing of chronic wounds, including chronic diabetic wounds.